(1) Field of the Invention The present invention relates to a recombinant poxvirus vaccine against feline herpesvirus type 1 (FHV-1). In particular the present invention relates to a recombinant raccoon poxvirus containing genes encoding a gB and/or gD precursor polypeptide from FHV-1.
(2) Prior Art
Feline viral rhinotracheitis (FVR) is caused by feline herpesvirus-1 (FHV-1). Feline herpesvirus (FHV-1), is a member of the genus alphaherpes virinae. This widespread virus is responsible for 40-45% of all respiratory infections of Felidae (the cat family). Most cats receiving veterinary care are vaccinated against this disease. Several existing modified-live (MLV) and inactivated vaccines (IV) against the disease have either residual virulence (MLV) or suffer from a lack of immunogenicity. Furthermore, none of the existing parenterally administered MLV or IV can protect vaccinated cats from infection with virulent virus (as opposed to the disease) when exposed to it. This automatically leads to latent infections, which are epidemiologically very important because of the ease and frequency by which latent FHV-1 is reactivated and spread by asymptomatic, latently infected carriers.
Several million domesticated cats that are kept as house pets in the U.S. receive annual vaccinations for FVR. The need for an improved vaccine is substantial.
The superior immunogenicity typically achieved with other vaccinia-based recombinants (Blacklaws, B., et al., Virology 177:727-736 (1990)) is an indicator that recombinants could be more effective and safer vaccines than existing preparations.
It has been previously demonstrated that there is a significant host immune response to viral glycoproteins during FHV-1 infection (Maes, R., et al., J. Virology 51:259-262 (1984)). The temporal development of immunity against FHV-1 glycoproteins in cats inoculated with FHV-1 on the oral, nasal and conjunctival mucosa has also been defined. Thus, the concurrent detection of virus-neutralizing antibody and glycoprotein-specific immunoprecipitins implied that FHV-1 glycoproteins were important in the induction of virus-neutralizing antibodies to FHV-1 in cats (Burgener, D. and Maes, R., American J. Vet. Res. 49:1673-1676 (1988); and Rota, P., et al., Virology 154:168-179 (1986)).
Over the last ten years, a large amount of information has accumulated concerning the immunity induced by the glycoproteins of the alphaherpesviruses HSV-1, PRV, EHV-1, MDV and BHV-1 and other herpesviruses (i.e. EBV, HCMV, HVS). The genome of herpes simplex virus-type 1 (HSV-1) codes for at least 10 antigenically distinct glycoproteins: gB, gC, gD, gE, gG, gH, gI, gJ, gK and gL (Spear, P., Glycoproteins specified by herpes simplex viruses. In "The Herpesviruses" (B. Roizman, ED.) Vol. 3 pp 315-356. Plenum, N.Y. (1984); and Hutchinson, L., et al., J. Virology 66: 2240-2250 (1992)). It has been established that these glycoproteins can be classified as either essential or nonessential for replication of the virus. Because of their biological role in virion adsorption and eggression from infected cells, viral glycoproteins are generally conserved throughout related subfamilies. Based on extensive work with HSV-1 and the animal herpesviruses, it has been defined that glycoproteins B and D are major immunogens, eliciting high titers of virus neutralizing (VN) antibodies and providing protective immunity in vaccinated animals against lethal challenge. So far, HSV-1 is the best model for the comparison of the immune response induced by various glycoproteins of a specific herpesvirus (Blacklaw, B., et al., Virology 177:727-736 (1990)). Individual HSV-1 glycoproteins (gB, gD, gH, gI, gE and gG) expressed in vaccinia virus were evaluated for their ability to (1) elicit neutralizing antibody titers, (2) increase the rate of HSV-1 clearance and (3) protect against lethal challenge and latency. Vaccinia recombinants expressing gB and gD were reported to be superior in eliciting high titers of VN-antibodies and full protection from the establishment of latency.
Glycoprotein B homologs have been mapped within the genomes of 14 herpesviruses: herpes simplex virus-1, herpes simplex virus-2, varicella-zoster virus, Epstein-Barr virus, human cytomegalovirus, equine herpesvirus-1, equine herpesvirus-4, bovine herpesvirus-1, bovine herpesvirus-2, pseudorabies virus, Marek's disease virus, herpesvirus saimiri, infectious laryngotracheitis virus and simian agent type 8 virus. This conservation is not surprising since gB, as well as glycoproteins D, H, K and L, have been shown to be essential for production of enveloped viruses (Spear, P., Glycoproteins specified by herpes simplex viruses. In "The Herpesviruses" (B. Roizman, ED.) Vol. 3 pp 315-356. Plenum, N.Y. (1984); Hutchinson, L., et al., J. Virology 66: 2240-2250 (1992); and MacLean, C., et al., J. Gen. Virology 72: 897-906 (1991)).
HSV-1 gB and also the gB homolog of PRV (gII) have been shown to form a dimeric protein on the surface of virions and infected cells. Furthermore, glycoprotein B has been implicated in the penetration of the host cell membrane and also in cell-to-cell spread of virus by fusion. It appears that a homolog gB in FHV-1 acts in the same way.
Glycoprotein D of HSV-1 has also been reported to be essential penetration of the nucleocapsid into susceptible cells (Fuller and Spear, J. of Virology 55:475-482 (1985); Spear et al., Herpes simplex virus:pathway of entry into cells. In "Cell Biology of Virus Entry, Replication and Pathogenesis" pp. 163-175 (1989); Johnson et al., J. of Virology 64: 2569-2576 (1990)). Although genes encoding gD homologs are generally conserved throughout herpesvirinae, VZV and the distal related herpesvirus, channel catfish herpesvirus do not contain gD homologs (Davison, A. and Scott, J., J. Gen. Virology 67: 1759-1816 (1986); Davison, A., Virology 186: 9-14 (1992)). Early studies with monospecific gD antisera or monoclonal antibodies have indicated that gD plays a role in virus penetration and cell fusion (Noble, A., et al., Virology 129: 218-224 (1983)). In one study by (Johnson, D., et al., J. of Virology 62: 4605-4612 (1988)) UV-inactivated (gD+) virions were reported to block the entry of WT-HSV-1 or HSV-2 into cells, whereas UV-inactivated virions which are phenotypically gD- were unable to block WT-HSV-1 or HSV-2 entry. Furthermore, mutant (gD-) virions were shown to be able to adsorb to cellular membranes but could not penetrate into the cells.
Besides their biological significance, these two glycoproteins are the major immunodominant polypeptides of herpesviruses, capable of the induction of protective immunity. Of all the HSV-1 glycoproteins, only antibodies to glycoprotein D and B can crossreact with the two types of simplex viruses (Marchioli, C., et al., J. of Virology 61: 3977-3982 (1987)). It has also been demonstrated that gD of HSV-1 induces the most potent monoclonal antibodies with the highest affinity for the HSV-1 virion (Para, M., et al., J. of Virology 55: 483-487 (1985); Iglesias, G., et al., Vet. Micro. 24: 1-10 (1990)). Furthermore, anti-gD monoclonal antibodies have been routinely generated from animals immunized with crude virion preps of HSV-1.
There is good evidence that glycoprotein B is as important an immunogen as gD. In HCMV seropositive individuals, for example, 40-70% of total virus-neutralizing activity in serum has been reported to be directed against gB (Britt, W., et al., J. of Virology 64: 1079-1085 (1990)). Such a preferential reactivity of human sera for a single virion component is unique, due to the fact herpesviruses contain many glycoproteins.
Both glycoproteins D and B of HSV-1, PRV and EHV-1 have been reported to protect mice from lethal challenge (Long, D., et al., Infection and Immunity 37:761-763 (1984)). In one study, mice immunized with gD, affinity-purified from cells infected with either HSV-1 or HSV-2, were protected from a lethal intraperitoneal (i.p) challenge by virus of either serotype (Eisenberg, R., et al., J. of Virology 56: 1014-1017 (1985)). Similarly, gp50 of pseudorabies virus, the gD homolog in the suid herpesvirus, has be reported to elicit VN-antibodies (Eloit, M., et al., J. of Gen. Virology 71:2425-2431 (1990)) and when expressed in vaccinia virus or Chinese hamster ovary cells, (Marchioli, C., et al. J. of Virology 61: 3977-3982 (1987)) protected immunized mice or rabbits from virulent challenge with PRV. In addition, a recombinant gp50 protects pigs, the natural host, from lethal challenge (Marchioli, C., et al. J. of Virology 61: 3977-3982 (1987); Riviere, M., et al., J. of Virology 66: 3424-3433 (1992)). Likewise, protection of mice immunized with recombinant adenoviruses expressing glycoprotein B of HSV-1, has also been demonstrated. Unlike in gD, correct glycosylation of gB appears to be essential for optimal immunogenicity. Mice immunized with recombinant gB isolated from mammalian cells, produced significantly higher titers of virus-neutralizing antibodies, when compared to animals immunized with recombinant gB isolate from procaryotes. An enhanced level of protection from lethal challenge was also demonstrated in vaccinates receiving the glycosylated (eukaryotic) recombinant polypeptide. In a study by van Drunen littel-van den Hurk, S., et al., J. of Gen. Virology 71: 2053-2063 (1990)), deglycoslyation of gI(gB) of BHV-1 resulted in a significant decrease in the production of serum neutralizing antibodies, due to modifications of three distinct carbohydrate containing continuous epitopes. Likewise, nonglycosylated HCMV gB produced in recombinant prokaryotic systems has been reported to be less immunogenic than the glycosylated protein produced in eukaryotes (Britt, W., et al., J. of Virology 64: 1079-1085 (1990)). In contrast, nonglycosylated forms of glycoprotein D, for example gIV of BHV-1, stimulate neutralizing antibodies at levels similar to those elicited by glycosylated forms. This comes as no surprise, since the nucleotide sequence of gp50 (gD) of PRV lacks potential N-linked glycosylation sites (Petrovskis, E., et al., J. of Virology 59: 216-223 (1986)). Recently, gD of HSV-1 has been expressed at high levels in baculoviruses. Although the recombinant protein was slightly smaller than the gD in HSV-1 infected Vero cells, due to differences in the glycosylation pattern of the two cell lines, the expressed protein was present on the membranes of SF9 cells and reacted with gD specific antibodies. Vaccination with the expressed protein resulted in the production of neutralizing antibodies to HSV-1 and complete protection against lethal HSV-1 challenge (Ghiasi, H., et al., Arch. Virology 121: 163-177 (1991)).
Because of these results, gD and gB of HSV-1 are the prime candidates for a subunit vaccines. The genes encoding gD and gB of various herpesviruses have been expressed in both prokaryotic and mammalian cells. Studies on mammalian cells expressing native and truncated gD polypeptides, along with synthetic peptides and V8 protease digestion products have enabled researchers to map its immunologically important continuous and discontinuous epitopes. Synthetic peptides representing one continuous epitope (amino acids 9-21) of gD(HSV-1) conjugated to ovalbumin or BSA, were reported to elicit high titers of antipeptide neutralizing antibodies in mice after immunization with adjuvants. Resistance to lethal challenge was also demonstrated in synthetic peptide-immunized mice (Eisenberg, R., et al., J. of Virology 56: 1014-1017 (1985)).
From the above, it is clear that humoral immunity to gD and gB appears to be a significant contributor to the clearance of the virus. However, this type of immunity is primarily important during the initial infection. Overall, cell-mediated immunity (CMI) appears to be more important. Not only is it essential in the acute phase of a herpesvirus infection but is also involved in virus clearance following reactivation or reinfection. The importance of CMI in resistance to HSV-1 is apparent by the fact that 80-90% of immunosuppressed patients have a high incidence of recurrence (Bernstein, D., et al., J. of Immunology 146:3571-3577 (1991)). Supporting the role of cell-mediated immunity are numerous reports of adoptive transfer experiments, conferring resistance to lethal HSV challenge. In a study by (Rooney, J., et al., J. of Virology 62: 1530-1534 (1988)), vaccinia recombinants containing the gD(HSV-1) gene under the control of an early vaccinia promoter were reported to elicit a better T-cell response than recombinants in which gD expression is controlled by a late vaccinia promoter. Both recombinant viruses produced potent neutralizing antibodies and protected immunized mice from both lethal HSV-1 challenge and latency establishment by the challenge virus for at least 6 weeks after immunization (Rooney, J., et al., J. of Virology 62: 1530-1534 (1988); Wachsman, M., et al., J. Gen. Virology 70:2513-2520 (1989); Wachsman, M., et al., J. of Inf. Dis. 159: 625-634 (1989)). However, reimmunization with the recombinants containing the early vaccinia promoter/gD construct resulted in a significant increase in neutralizing antibody titers lasting over 1 year. Vaccinia recombinants containing the late vaccinia promoter/gD gene fusion failed to protect from cutaneous disease following administration of a high dose of HSV-1. Protection against cutaneous lesions is associated with the induction of HSV-1 specific T-cell responses. Furthermore, proliferation of lymph node cells in response to HSV-1 antigens was demonstrated only in mice immunized with the Vac(early promoter)/gD- and not Vac(late promoter)/gD-constructs. It appears that temporal expression of glycoprotein genes in antigen presenting cells is important in the induction of immunity to herpes viral disease (Wachsman, M., et al., (1989)).
Additional evidence for the role of these glycoproteins in cell-mediated immunity response comes from studies involving immunized mice transplanted with cells expressing herpesvirus glycoproteins. Nakagama, H., et al., FASEB J. 5: 104-108 (1991) reported significant differences in lymphocyte infiltration and antigen clearance in syngeneic unimmunized mice, transplanted with (HSV-1) gD-transfected BALB/3T3 cells, as compared to mice immunized with HSV-1. In the later case, the transfected cells elicited massive lymphocyte infiltration of mainly THY1+ and CD8+ lymphocytes along with a small number of CD5+, CD4+, and B-lymphocytes in the HSV-1- immunized mice. In contrast, in unimmunized mice, little evidence of cellular infiltration could be detected and transplanted cells could be detected for as long as 7 days. In immunized animals however, the transplanted cells were mostly destroyed by day 4, despite the presence of anti-HSV-1 antibodies at the time of transplantation. Likewise, cells from the spleen and lymph nodes of gB-immunized mice have been reported to protect syngeneic mice against lethal challenge.
It is generally believed that reactivation of latent herpesvirus occurs more frequently than episodes of recurrent disease. Administration of gD or gB to latently infected animals, reduces the frequency of reactivation, the severity of recurrent disease and the duration of shedding (Bernstein, D., et al., J. of Immunology 146: 3571-3577 (1991)). In guinea pigs latently infected with HSV-2, the adoptive transfer of clones expressing either glycoprotein D or B, significantly reduced the number and severity of subsequent symptomatic recurrent infections with a concomitant reduction in cervicovaginal HSV-2 shedding. In this study the author concluded that the reduction in clinical disease was the result of lymphokine activated cellular immunity in which the transfer of HSV-1 gD or gB into latently infected animals resulted in the production of other cytokines by HSV-1 sensitized T-cells. This could further increase critical responses, such as natural killer cells, needed for the clearance of the reactivated virus. Further evidence for the involvement of lymphokine activity in CMI elicited by herpesvirus glycoproteins was provided by (Zarling, J., et al., J. of Immunology 136: 4669-4673 (1986)). Administration of gD or gB, expressed in mammalian cells to HSV-1 seropositive individuals stimulated proliferation of their peripheral blood lymphocytes and interleukin-2 production by these cells. Interestingly, IL-2 can also significantly enhance cellular and humoral immunity in cows when included in either a gD subunit or MLV-vaccine (Reddy, P., et al., Vet. Immuno. Immunopath. 23: 62-74 (1989); Hughes, H., et al., Immunology 74:461-466 (1991)). Likewise, high antibody responses and cell mediated immunity to HSV-1 were recently reported in mice immunized with a recombinant expressing a glycoprotein D/Interleukin-2 fusion protein (Hinuma, S., et al., FEBS 288, 138-142 (1991)).
Objects
It is an object of the present invention to provide an approach for vaccination of cats against feline herpesvirus type 1 (FHV-1) using a recombinant vaccine that corrects the shortcomings of currently available commercial vaccines which lack immunogenicity, have an inability to prevent infection, have adjuvant induced side effects, have a potential reversion to virulence, or have a residual virulence.
It is further an object of the present invention to provide the recombinant viral vaccines expressing a single FHV-1 viral glycoprotein or two (2) glycoproteins.
It is further an object to provide novel live recombinant vaccines which express gB and gD of FHV-1 in a recombinant poxvirus vector that is safe for use in cats and which provides immunogenicity in cats.
It is particularly an object to provide recombinant raccoon poxviruses expressing the FHV-1 gB and/or gD genes.
Further, it is an object to immunize cats with recombinant raccoon poxviruses, which when exposed to virulent FHV-1, provide superior protection against clinical signs and shedding of the virulent virus.
These and other objects will become increasingly apparent by reference to the following description and the drawings.